3'-(2')-amino or thiol-modified, fluorescent dye-coupled oligonucleotides and a process for the preparation and the use thereof

• US 5,659,025 A
• Filed: 06/01/1995
• Issued: 08/19/1997
• Est. Priority Date: 12/11/1990
• Status: Expired due to Fees

• Current Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Original Assignee: Hoechst Aktiengesellschaft (Sanofi )
• Inventors: Konrad, Renate, Herrlein, Mathias, Engels, Joachim, Mag, Matthias
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/457,377
• Time in Patent Office: 810 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.33, 536/25.34, 536/25.5, 536/25.32, 435/6
• US Class Current: 536/23.1
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

The OH group located in the 3'"'"' and/or 2'"'"' position of a nucleoside, nucleotide or oligonucleotide is derivatized to an amino or thiol group and subsequently a fluorescent dye is coupled thereto. The resulting 3'"'"'- and/or 2'"'"'-amino- and thiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of complementary strands in the presence of a template strand or of oligonucleotides and for the detection of genetic material. They have the advantage that the fluorescent label need no longer be attached to the 5'"'"' end of the oligonucleotide or to the nucleobase, and thus need not be introduced during the chemical synthesis as in labeling techniques hitherto known, while the known and conventional methods have the disadvantage that only a few polymerases can be employed for the synthesis, the acceptance of the triphosphates by the polymerases diminishes and, moreover, a large substrate excess is necessary.

Claims

1. A method for the synthesis of labeled 3'"'"'-amino- and thiol-modified DNA and RNA oligonucleotide strands which comprises

synthesizing the strand using a suitable primer, a template strand and a polymerase in the presence of four nucleoside triphosphates, and

terminating the synthesis with a 3'"'"'-(2'"'"')amino-modified nucleotide compound of the formula I ##STR13## in which R¹ is a purine or pyrimidine base, wherein R¹ can in each case be identical or different,

R² is hydrogen, a hydroxyl, a protected hydroxyl or methoxy group in α

- or β

-position,

R³ is a fluorescent dye in the α

or β

-position bonded via an amino or thiol group,

n is a number greater than or equal to zero and less than 3000,

R⁴ is a 5'"'"' protective group or phosphate, pyrophosphate or triphosphate,

R⁵ is oxygen,

R⁶ is a hydroxyl, methoxy group or hydrogen in α

- or β

-position, where R⁶ can in each case be identical or different, and

X and Y are oxygen or sulfur, where X and Y can in each case be identical or different in the synthesis.

2. A method for microscopic and macroscopic fluorescence detection in vivo and in vitro of labeled oligonucleotides synthesized according to the method of claim 1.

3. The method according to claim 1, wherein said synthesis of labeled DNA and RNA oligonucleotides is carried out in the presence of the complement of said DNA and RNA oligonucleotides.